Dissecting Complexity 2: Microfilaments

1
Dissecting Complexity 2Microfilaments

• How actin-based polymers contribute to cellular
  structure, behavior, and motility

2
An overview of the actin cytoskeleton in a
cultured epithelial cell, using antibodies and
immuno- fluorescence. Actin is at the cell
cortex and also organized into fibers that extend
through the cell.
3
Formation of microfilaments (MFs) from the
protein actin
4
The functional complexity of MFs and
their Associated proteins
5
Actin at the edge of a cultured cell, looking at
all actin (top) or only recently added actin
(bottom). Actin polymerizes at the cell
periphery
6
Direct evidence for the addition of actin
monomers to the membrane-end of MFs
7
Actin polymerization is controlled, in part, by
an initiating complex that includes actin-like
proteins
8
The Arp2/3 complex not only initiates
MF polymerization, it can bind to the walls
of existing MFs, promoting the formation
of branches, which turn that region of the
cell into a gel (as opposed to a sol).
9
Actin polymerization is also regulated by small
proteins that bind to soluble actin and modify
its behavior in solution. Here is profilin,
which catalyzes the exchange of ADP for ATP in
the actin cleft, increasing the pool of
polymer- ization-competent monomer
10
There are also proteins that retard MF growth
While profilin enhances MF growth by
increasing the concentration of
ATP-actin, Thymosin reduces it by making
a complex that will not polymerize
11
Diagramatic representation of the pathways The
help to regulate MF formation
12
(No Transcript)
13
So how does a complex precess like this actually
work?

• Amoeboid motility does not require MTs
• It is immediately poisoned by drugs that block
  actin polymerization, like Cytochalesins D and E
  or Latrunculin A
• It fails in several mutant strains that lack key
  actin assembly proteins (though it is amazingly
  robust to mutation)
• Infer, we need to understand the controll of
  actin polymerization and organization

14
Like MTs, MFs can include associated
proteins that alter the properties of the
polymer. Cofilin binds to F-actin and distorts
the polymer, making it less stable.
15
Capping proteins, like Cap-Z can silence one MF
end for further subunit addition in this case it
is the fast-growing (plus) end that is turned
off.
16
Proteins that bind to the walls of MFs can
bind them together in different geometries
17
Four proteins that bind the sides of MFs
and bundle them into different geometries
18
Diagrams of examples of MF bundling
19
TEM image and diagram of MF Bundling in a
microvillus
20
Diagram of MF web formed when cross-linking is
done by the long, fibrous protein, Filamin
21
When filamin is under-expressed, due to mutation,
cell morphology and motility are abnormal.
Cells depleted for Cell
expressingfilamin by LOF mutation normal
filamin levels
22
Diagram of the consequences of wall-binding by a
different MF-associated protein, Gelsolin, which
distorts and breaks the MF.
23
Gelsolin and proteins like it can break up
MF either in vitro (as shown here) or in vivo,
leading to rapid reshaping of the actin
cytoskeleton
24
Breaks in MFs mean more shorter MFs and
therefore more ends (for a given amount
of polymer. Thus, both growth and shrinkage of
polymer can be faster after MF severing.
25
Blood platelets (thrombocytes) looking as they do
in circulating blood (left), after attachment to
a surface (center) and during a
platelet reaction, where they attach to a
substratum and contract.
26
(No Transcript)
27
The formation of filopodia Is like the formation
of a labile microvillus. The organellar
structure is defined by non-covalent bonds
between proteins that hold them together in a
minimum energy association. The question is,
what initiates the polymerization of the MFs?
28
Formins are now recognized as the molecules that
can initiate MF polymerization at the PM and
allow continued addition of actin at the PM while
an association with the membrane is maintained

• Formins contain conserved domains, the
  Formin-homology (FH) domains. FH1 and 2 define a
  comparatively short polypeptide that can serve as
  an efficient nucleator of MF polymerization

29
What is Pyrene actin and how does it help to
measure MT formation? What is phalloidin and why
does rhodamine Phalloidin show up actin in
cells? What is a Gal1 promoter, and why would one
use it for control of expression in vivo? Why
doesnt actin polymerize efficiently on its
own (Fig 2A)? Why does spectrin-actin act as a
seed? What is CB, and how does it block MF
growth How can the authors conclude that MFs
initiated by FH1/FH2 are adding actin at their
barbed (plus) ends?
30
Projection morphology and other actin-dependent
shape changes suggest that controlled
polymerization can also give rise to
unidirectional fibers, the filopodia

• Microvilli are of this form
• Dynamic projections from not only platelets but
  also neuronal growth cones are of this form
• Even projections from sperm and egg, involved in
  fertilization, are of this form
• There must be a membrane-associated actin
  initiator that does not involve branching